The long-term objective of this project is to understand the mechanisms of voltage-dependent ion channels. This study will focus on three voltage-dependent ion channels, including a eukaryotic voltage-dependent K+ channel (Aim 1), a prokaryotic voltage-dependent K+ channel (Aim 2), and a eukaryotic voltage-dependent H+ channel (Aim 3). X-ray crystallography will be used to determine atomic structures of these membrane proteins and electrophysiological recordings and ion flux assays will be used to analyze the function. In addition, NMR spectroscopy will be used to study protein structure, dynamics, and lipid interactions. Work funded by this research proposal has already led to an understanding of the open conformation structure of a voltage- dependent K+ channel. Closed conformations are still unknown. The goal of the first two aims is to determine structures of partially and fully closed conformations of an entire voltage-dependent channel (Aim 1) and an isolated voltage sensor (Aim 2) and to correlate these conformations with function so that we can understand how membrane voltage controls the conformation of voltage sensors and voltage-dependent ion channels. The closed conformations will be induced through specific mutations that are identified and evaluated in electrophysiological assays. In the third aim we seek to determine a first atomic structure of a voltage- dependent H+ channel through x-ray crystallography. Because the H+ is invisible to x-rays we will use NMR spectroscopy to study the interaction of the H+ channel with its substrate. Voltage-dependent ion channels produce electrical signals in living cells. They are essential to nervous system as well as skeletal, cardiac and smooth muscle function. Certain voltage-dependent ion channels are targets of action for important pharmacological agents. An in-depth understanding of the atomic structure and mechanisms of voltage- dependent ion channels holds promise for new approaches in the future to treat diseases such as epilepsy and cardiac arrhythmia. PUBLIC HEALTH RELEVANCE: Life has evolved a marvelous molecular system for producing electrical signals that underlie our thoughts and movements. The molecules in this system are known as voltage-dependent ion channels. This project seeks to understand the atomic basis of voltage-dependent ion channel function with the hope that this knowledge will lead to new therapies for diseases such as epilepsy and cardiac arrhythmia.